The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae

Abstract

Backgroundl-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains.ResultsChemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (Km = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10−3 and 1.8 g L−1, respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L−1 l-arabinose and 20 g L−1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2.ConclusionIts high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1047-6) contains supplementary material, which is available to authorized users.

Laboratory evolution of a glucose-phosphorylation-deficient, arabinose-fermenting S. cerevisiae strain reveals mutations in GAL2 that enable glucose-insensitive l-arabinose uptake

“…Expression of the high-affinity l -arabinose transporter Pc AraT from P. chrysogenum enables l -arabinose uptake in the presence of glucose (Bracher et al. 2018 ). To simultaneously abolish the ability to metabolize d -glucose, an expression cassette for Pc AraT (Bracher et al 2018 ) was integrated into the HXK2 locus.…”

“… 2018 ). To simultaneously abolish the ability to metabolize d -glucose, an expression cassette for Pc AraT (Bracher et al 2018 ) was integrated into the HXK2 locus. Subsequent introduction of a functional URA3 allele from S. cerevisiae CEN.PK113–7D (Additional File 3d) yielded the prototrophic ‘arabinose specialist’ S. cerevisiae strain IMX728 ( hxk1Δ glk1Δ gal1Δ:: { Spcas9 -amdSYM} gre3Δ:: {NPPP} hxk2Δ::PcaraT gal80Δ ::{ARAP}).…”

“… 2015 ; Li et al. 2015 ; Bracher et al 2018 ). K m values of these heterologous transporters for l -arabinose ranged from 0.13 to 263 mM, while reported V max values ranged from 0.4 to 171 [nmol l -arabinose (mg biomass) −1 min −1 ].…”

“…K m values of these heterologous transporters for l -arabinose ranged from 0.13 to 263 mM, while reported V max values ranged from 0.4 to 171 [nmol l -arabinose (mg biomass) −1 min −1 ]. Several heterologous transporters, including the high-affinity l -arabinose proton symporter Pc AraT from P. chrysogenum (Bracher et al 2018 ) allowed for l -arabinose uptake in the presence of d -glucose (Subtil and Boles 2011 ; Knoshaug et al. 2015 ; Li et al.…”

Engineering of Pentose Transport in Saccharomyces cerevisiae for Biotechnological Applications

“…When expressed in a Larabinose-fermenting S. cerevisiae strain in which GAL2 was deleted, growth on L-arabinose could be restored. PcAraT is a H + :L-arabinose symporter with high-affinity for L-arabinose (K m = 0.13 mM) while D-xylose and D-glucose are not transported (Bracher et al, 2018).…”